Polyarginine-coated magnetic nanovector and methods of use thereof

ABSTRACT

Polyarginine-coated nanoparticle, and methods for making and using the nanoparticle. The nanoparticle can have a core that includes a material that imparts magnetic resonance imaging activity to the particle and, optionally, include one or more of an associated therapeutic agent, targeting agent, and diagnostic agent.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Patent Application No.61/646,101, filed May 11, 2012, expressly incorporated herein byreference in its entirety.

STATEMENT OF GOVERNMENT LICENSE RIGHTS

This invention was made with government support under Grant Nos.RO1CA134213 and RO1EB006043 awarded by the National Institutes ofHealth. The government has certain rights in the invention.

STATEMENT REGARDING SEQUENCE LISTING

The sequence listing associated with this application is provided intext format in lieu of a paper copy and is hereby incorporated byreference into the specification. The name of the text file containingthe sequence listing is 41027_SEQ_FINAL.txt. The text file is 631 Bytes;was created on May 7, 2013; and is being submitted via EFS-Web with thefiling of the specification.

BACKGROUND OF THE INVENTION

Small interfering RNAs (siRNAs) can silence gene expression in a highlyspecific manner for treating genetic disorders, signifying a newapproach in cancer therapy through the regulation of aberrant geneexpression inherent to cancer. However, the physicochemicalcharacteristics of siRNA (e.g., high molecular weight, anionic charge,and hydrophilic character) hinder its passive diffusion across cellmembranes precluding any therapeutic function. Furthermore, siRNAmolecules are highly vulnerable to degradation. Thus, for effectivesiRNA delivery, siRNA carriers are needed to protect siRNA, facilitatecellular entry, avoid endosomal compartmentalization, and promotelocalization in the cytoplasm where the siRNA cargo can be recognized bythe RNA-induced silencing complex (RISC). Inorganic nanoparticles (NPs)designed for this application are propitious as they can be engineeredfor simultaneous diagnostics and therapeutics (theranostics). Currently,many NP core material formulations such as gold, silica, semiconductors,and metal oxides are being evaluated as siRNA carriers (nanovectors).Among them superparamagnetic iron oxide NPs possess superiorphysicochemical and biological properties ideal for in vivo magneticresonance imaging (MRI) and drug delivery.

The success of nanovectors relies on the apt design and integration ofcoatings that ensure biocompatibility and stability in a biologic milieuand proper intracellular trafficking. To date, most nanovectorsdeveloped for gene delivery applications are coated with cationicsynthetic polymers (e.g., polyethylenimine (PEI), poly amidoamines(PAMAM)) or lipids. A common characteristic among these carriers istheir high cationic charge density at physiological pH, whichcontributes to both the complex formation with anionic siRNA andinteraction with the negatively charged cell membrane. This interactionwith cell membranes typically leads to the endocytosis of thenanovector, entrapping the nanovector within cellular endosomalvesicles. Within the cellular endosomes the amino groups of cationicpolymers function as proton sponges causing the swelling and eventualrupture of the endosome releasing the nanovector into the cytoplasm, aprocess known as endosomal escape. However, the high cationic chargedensity of these synthetic polymers also renders them highly cytotoxic.

Despite the advances in the development of siRNA carriers, a need existsfor an siRNA carrier that is effective intracellular delivery of siRNAand that provides a safer alternative to the highly cationicnanovectors. The present invention seeks to fulfill this need andprovides further related advantages.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a nanoparticle having apolyarginine coating. In certain embodiments, the nanoparticle has acore that includes a material that imparts magnetic resonance imagingactivity to the particle. The nanoparticle can further include one ormore of a therapeutic agent that can be delivered by the particle, atargeting agent to target the nanoparticle to a site of interest, and adiagnostic agent that allows for imaging of the particle.

In one embodiment, the nanoparticle, comprises:

(a) a core having a surface and comprising a core material; and

(b) a polyarginine coating covalently coupled to the surface of thecore.

In certain embodiments, the nanoparticle further comprises a therapeuticagent covalently coupled to the nanoparticle. In other embodiments, thenanoparticle further comprises a targeting agent covalently coupled tothe nanoparticle. In further embodiments, the nanoparticle furthercomprises a therapeutic agent and a targeting agent covalently coupledto the nanoparticle.

Suitable therapeutic agents include small organic molecules, peptides,aptamers, proteins, and nucleic acids. In certain embodiments, thetherapeutic agent is an RNA (e.g., siRNA) or a DNA. In certainembodiments, the therapeutic agent is covalently coupled to the coatingis coupled through a cleavable linkage.

Suitable targeting agents include small organic molecules, peptides,aptamers, proteins, and nucleic acids.

In certain embodiments, the nanoparticles of the invention furthercomprise a diagnostic agent. Representative diagnostic agents includeoptical agents, such as fluorescent agents.

In certain embodiments, the nanoparticle's core material is a magneticmaterial.

In another aspect, the invention provides a composition comprising ananoparticle of the invention and a carrier suitable for administrationto a warm-blooded subject.

In a further aspect of the invention, a method for introducing ananovector into a cell via transcytosis is provided. In one embodiment,the method includes contacting a cell with a nanoparticle of theinvention.

In another aspect of the invention, a method for silencing or reducingthe expression level of a gene is provided. In one embodiment, themethod includes contacting a cell of interest with a nanoparticle of theinvention.

In another aspect, the invention provides a method for treating atissue. In one embodiment, the method includes contacting a tissue ofinterest with a nanoparticle of the invention.

In a further aspect of the invention, a method for silencing or reducingthe expression level of a gene is provided. In one embodiment, themethod includes contacting a cell of interest with a nanoparticle of theinvention.

In another aspect, the invention provides a method for detecting cellsor tissues by magnetic resonance imaging. In one embodiment, the methodincludes:

(a) contacting cells or tissues of interest with a nanoparticle of theinvention; and

(b) measuring the level of binding of the nanoparticle, wherein anelevated level of binding, relative to normal cells or tissues, isindicative of binding to the cells or tissues of interest.

DESCRIPTION OF THE DRAWINGS

The foregoing aspects and many of the attendant advantages of thisinvention will become more readily appreciated as the same become betterunderstood by reference to the following detailed description, whentaken in conjunction with the accompanying drawings.

FIGS. 1A-1D illustrate chemical schemes for synthesis of representativemagnetic nanovectors of the invention. FIG. 1A is a schematicillustration of a representative amidated PEG-passivated iron oxidenanoparticle (NP) useful for making representative transfection vectorsof the invention. FIG. 1B illustrates the chemical structures and thepKa's of cationic polymers pLys, pArg, and PEI used to functionalize theNP. FIG. 1C illustrates the covalent coupling of the cationic polymer toNP. to provide the cationic polymer coated nanoparticle. FIG. 1Dillustrates the coupling of the fluorophore-modified siRNA to thecationic polymer coated nanoparticle to provide a representativemagnetic nanovector of the invention.

FIGS. 2A-2C illustrate the proton NMR spectra of representativenanovectors verifying cation polymer coating attachment. FIG. 2Acompares the spectra of NP, PEI, and NP-PEI. FIG. 2B compares thespectra of NP, pLys, and NP-pLys. FIG. 2C compares the spectra NP, pArg,and NP-pArg. All samples were analyzed in D₂O.

FIGS. 3A-3D provides the characterization of representative nanovectors.FIG. 3A compares gel retardation assay results for representativeevaluating covalent attachment of siRNA to NPs under normalelectrophoresis conditions (Normal Gel) and under heparin treatment(Heparin Gel) to disrupt electrostatic interactions between cationic NPsand anionic siRNA. FIG. 3B compares Z-average hydrodynamic sizes ofrepresentative nanovectors before and after siRNA attachment. FIG. 3Ccompares zeta potentials of representative nanovectors before and aftersiRNA attachment. FIG. 3D compares TEM images of representativenanovectors at two magnifications (scale bars correspond to 10 nm).

FIGS. 4A and 4B compare titration curves evaluating the bufferingcapacity (addition of sodium hydroxide) of each nanovector formulation.FIG. 4A compares the titration curves for representative nanovectors.FIG. 4B compares the titration curves for the corresponding cationicpolymers.

FIGS. 5A and 5B illustrate the magnetic properties of nanovectors. FIG.5A compares R2 maps of gel phantoms containing representativenanovectors at different concentrations (mM). FIG. 5B compares R2relaxivity of representative nanovectors as a function of Feconcentration were used to determine relaxivities, which yieldedrelaxivity values of 78.2 mM⁻¹S⁻¹ for NP-PEG-siRNA, 103.7 mM⁻¹S⁻¹ forNP-pArg-siRNA, 131.8 mM⁻¹S⁻¹ for NP-pLys-siRNA, and 113.6 mM⁻¹S⁻¹ forNP-PEI-siRNA.

FIGS. 6A-6I compare siRNA delivery, cell viability, and GFP knockdownfor representative nanovectors. Relative amount of siRNA delivered toC6/GFP⁺ (FIG. 6A), MCF7/GFP⁺ (FIG. 6B), and TC2/GFP⁺ (FIG. 6C) cells bynanovectors of three different formulations. Influence of nanovectortreatments on cell viability of C6/GFP⁺ (FIG. 6D), MCF7/GFP⁺ (FIG. 6E),and TC2/GFP⁺ cells (FIG. 6F) (viability was normalized to untreatedcells). Efficiency of nanovector treatments on silencing GFP expressionin C6/GFP⁺ (FIG. 6G), MCF/GFP⁺ (FIG. 6H), and TC2/GFP⁺ (FIG. 6I) cells(GFP expression was normalized to untreated cells).

FIGS. 7A-7C are confocal fluorescence microscopy images evaluating siRNAinternalization and subsequent GFP knockdown for representativenanovectors. Images were acquired from C6/GFP+ (FIG. 7A), MCF7/GFP+(FIG. 7B), and TC2/GFP+ (FIG. 7C) cells 48 hours post treatment withNP-pArg-siRNA, with untreated cells as a reference. Scale barcorresponds to 20 μm.

FIGS. 8A and 8B are TEM images of C6/GFP⁺ cells treated with threerepresentative nanovector formulations (NP-pLys-siRNA, NP-pArg-siRNA,and NP-PEI-siRNA). FIG. 8A illustrates internalization of nanovectors.FIG. 8B illustrates intracellular localization of nanovectors. Scalebars represent 250 nm.

FIG. 9 is a TEM image of NP-PEG-amine (NP) useful for preparingrepresentative nanovectors of the invention. Scale bar corresponds to 10nm.

FIG. 10 is a schematic illustration of the preparation a nanoparticlehaving a modified surface (NP-SAS-PEG-NH₂) useful for preparingrepresentative nanovectors of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a nanoparticle having a polyargininecoating. In certain embodiments, the nanoparticle has a core thatincludes a material that imparts magnetic resonance imaging activity tothe particle. The nanoparticle can further include one or more of atherapeutic agent that can be delivered by the particle, a targetingagent to target the nanoparticle to a site of interest, and a diagnosticagent that allows for imaging of the particle. The therapeutic,targeting, and diagnostic agents can be covalently coupled to thenanoparticle. Methods for making and using the nanoparticles are alsoprovided.

Nanoparticle Having Polyarginine Coating

In one aspect, the invention provides a multifunctional nanoparticlehaving a polyarginine (pArg) coating. In one embodiment, thenanoparticle comprises (a) a core having a surface and comprising a corematerial and (b) a polyarginine coating covalently coupled to thesurface of the core.

As used herein, the phrase “core having a surface and comprising a corematerial” refers to a solid nanoparticle. The nanoparticle core is nothollow (e.g., not a solid shell encapsulating a void). The core materialcan impart functional properties to the nanoparticle (e.g., magneticproperties). The core material is not a polymeric material (e.g., thenanoparticle is not a polymer nanoparticle or a polymeric nanosphere).As used herein the term “polymeric material” refers to an organicpolymer material (e.g., poly(glycidyl methacrylate), poly(styrene),poly(alkylacrylate)). The core's surface defines the core's outermostsurface. In the practice of the invention, the core's surface ischemically modified to have a coating thereon. In certain embodiments,the nanoparticle core is a solid core comprising a material havingmagnetic resonance imaging activity (e.g., iron oxide).

The phrase “polyarginine coating covalently coupled to the surface ofthe core” refers to coating that substantially surrounds the core andthat is covalently coupled to the core's surface. The coating can bedirectly covalently coupled to the core surface or covalently coupled tothe core's surface through one or more other materials (e.g., layersintermediate the core surface and polyarginine coating) that arecovalently coupled to the core's surface. The term “polyargininecoating” refers to a coating that is prepared by covalently couplingpolyarginine to the core's surface or by covalently couplingpolyarginine to a material that is covalently coupled to the core'ssurface. The nature of the covalent coupling of polyarginine to thecore's surface to provide the coating is not particularly critical.Polyarginine can be covalently coupled to the surface by any one of avariety of chemistries known to the skilled person.

In certain embodiments, the nanoparticle of the invention, in additionto the polyarginine coating, includes a poly(alkylene oxide) oligomercoupled to the nanoparticle core through a siloxane layer. In thisembodiment, the polyarginine is covalently coupled to the poly(alkyleneoxide) oligomer (e.g., poly(ethylene oxide) oligomer). A representativeprocess for covalently coupling polyarginine to a nanoparticle corehaving poly(ethylene oxide) oligomers coupled thereto is illustrated inFIGS. 1A and 1C. Referring to FIG. 1A, the poly(ethylene oxide) oligomercovalently coupled to the core has a terminal amino (—NH₂) group that isfunctionalized to provide a reactive group (i.e., iodoacetamide)suitable for reaction with a complementary reactive group (i.e., thiol)introduced into the polyarginine (see FIG. 1C). Reaction provides thenanoparticle having a polyarginine coating. In this embodiment, thenanoparticle comprising a poly(alkylene oxide) oligomer intermediate thecore and the polyarginine coating. As illustrated in FIG. 1D, thenanoparticle is further modified to include an siRNA (illustrated asincluding a fluorescent agent coupled thereto) by further suitablefunctionalization of the nanoparticle and siRNA. The terminal aminogroups of the poly(ethylene oxide) oligomer serve as points forfunctionalization of the nanoparticle. Polyarginine, therapeutic agents(e.g., siRNA), targeting agents, and diagnostic agents can be covalentlycoupled to the nanoparticle through these terminal amino groups by avariety of chemistries known to those of skill in the art.

As illustrated in FIG. 1A, in certain embodiments, the poly(alkyleneoxide) oligomer is covalently coupled to the core through intermediatesiloxane linkages. The siloxane layer forms a coating on the coresurface. The siloxane is anchored to the core surface (e.g., oxidesurface) by interactions between the core surface and functional groupsof the siloxane (i.e., to provide core-siloxane linkages). In thisembodiment, amino-functionalized poly(alkylene oxide) oligomer isreacted with a suitably functionalized siloxane layer (e.g., carboxylicacid groups). As shown in FIG. 1A, the siloxane layer is covalentlycoupled to the nanoparticle core and the amino-functionalizedpoly(ethylene oxide) is covalently coupled to the siloxane layer byamide linkages.

The representative nanoparticle (NP) illustrated in FIG. 1A that isuseful for making the functionalized nanoparticles of the invention hasa core having a radius from about 5 to about 20 nm (e.g., about 6 nm), asiloxane layer having a radius from about 1 to about 5 nm (e.g., about 2nm), and a poly(alkylene oxide) layer having a radius from about 2 toabout 15 nm (e.g., about 12 nm). The core of the nanoparticle isrelatively small (e.g., 5 to 20 nm; 10-15 nm) and monodispersed.

In certain embodiments, the number of amino groups per nanoparticleprior to reaction to provide the siloxane coated nanoparticle is fromabout 30 to about 200. In one embodiment the nanoparticle includes about70 amino groups. The nanoparticles of the invention include from about0.1 to about 0.8 percent by weight polyarginine per nanoparticle. Theweight ratio of nanoparticle to siRNA is from about 5 to about 20(defined as Fe mass of NP: siRNA mass). In certain embodiments, thepolyarginine and the siRNA are coupled to the amino groups provided bythe amino-polyethylene oxide (PEG-amino). Targeting and/or diagnosticagents can also be covalently coupled to the nanoparticle through theseamino groups.

As described herein, the nanoparticles of the invention advantageouslyinclude a polyarginine coating. As noted above, the polyarginine coatingis prepared by covalently coupling polyarginine to the nanoparticle coreor by covalently coupling polyarginine to a material that is covalentlycoupled to the core. Suitable polyarginines have a molecular weight fromabout 2,000 to about 200,000 grams/mole. In certain embodiments, thepolyarginine has a molecular weight from about 5,000 to about 100,000grams/mole. In other embodiments, the polyarginine has a molecularweight from about 10,000 to about 50,000 grams/mole. In furtherembodiments, the polyarginine has a molecular weight from about 7,500 toabout 15,000 grams/mole.

In certain embodiments, in addition to the polyarginine coating, thenanoparticle of the invention includes a poly(ethylene oxide) oligomercoupled to the nanoparticle core through a siloxane layer. Suitablepoly(ethylene oxide) oligomers include poly(ethylene oxides) (PEO orPEG) and poly(ethylene oxide) copolymers such as block copolymers thatinclude poly(ethylene oxide) and poly(propylene oxide) (e.g., PEO-PPOand PEO-PPO-PEO). In one embodiment, the poly(ethylene oxide) oligomeris a poly(ethylene oxide). In certain embodiments, poly(ethylene oxide)oligomer has a molecular weight (weight average, Mw) of from about 0.3to about 40 kDa. In others embodiments, the poly(ethylene oxide)oligomer has a molecular weight of from about 1.0 to about 10 kDa. Incertain embodiments, the poly(ethylene oxide) oligomer has a molecularweight of about 10 kDa.

The nanoparticle includes a core material. For magnetic resonanceimaging applications, the core material is a material having magneticresonance imaging activity (e.g., the material is paramagnetic). Incertain embodiments, the core material is a magnetic material. In otherembodiments, the core material is a semiconductor material.Representative core materials include ferrous oxide, ferric oxide,silicon oxide, polycrystalline silicon oxide, silicon nitride, aluminumoxide, germanium oxide, zinc selenide, tin dioxide, titanium, titaniumdioxide, nickel titanium, indium tin oxide, gadolinium oxide, stainlesssteel, gold, and mixtures thereof.

The particle of the invention has nanoscale dimensions. Suitableparticles have a physical size less than about 50 nm. In certainembodiments, the nanoparticles have a physical size from about 10 toabout 50 nm. In other embodiments, the nanoparticles have a physicalsize from about 10 to about 30 nm. As used herein, the term “physicalsize” refers the overall diameter of the nanoparticle, including core(as determined by TEM) and coating thickness. Suitable particles have amean core size of from about 2 to about 25 nm. In certain embodiments,the nanoparticles have a mean core size of about 7 nm. As used herein,the term “mean core size” refers to the core size determined by TEM.Suitable particles have a hydrodynamic size less than about 150 nm. Incertain embodiments, the nanoparticles have a hydrodynamic size fromabout 20 to about 150 nm. In certain embodiments, the nanoparticles havea hydrodynamic size of about 33 nm. As used herein, the term“hydrodynamic size” refers the radius of a hard sphere that diffuses atthe same rate as the particle under examination as measured by DLS. Thehydrodynamic radius is calculated using the particle diffusioncoefficient and the Stokes-Einstein equation given below, where k is theBoltzmann constant, T is the temperature, and η is the dispersantviscosity:

$R_{H} = {\frac{k\; T}{6\pi \; \eta \; D}.}$

A single exponential or Cumulant fit of the correlation curve is thefitting procedure recommended by the International StandardsOrganization (ISO). The hydrodynamic size extracted using this method isan intensity weighted average called the Z average.

The nanoparticles of the invention include the polyarginine-coatednanoparticles described above that further include one or more otheragents. Thus, in other embodiments, the nanoparticles of the inventionfurther include one or more of a therapeutic agent that can be deliveredby the particle, a targeting agent to target the nanoparticle to a siteof interest, or a diagnostic agent that allows for imaging of theparticle. The therapeutic, targeting, and diagnostic agents can becovalently coupled to the nanoparticle.

Therapeutic Agents.

Therapeutic agents effectively delivered by the nanoparticles of theinvention include small organic molecules, peptides, aptamers, proteins,and nucleic acids. In certain embodiments, the therapeutic agent is anRNA or a DNA (e.g., an siRNA).

Suitable therapeutic agents include conventional therapeutic agents,such as small molecules; biotherapeutic agents, such as peptides,proteins, and nucleic acids (e.g., DNA, RNA, cDNA, siRNA); and cytotoxicagents, such as alkylating agents, purine antagonists, pyrimidineantagonists, plant alkaloids, intercalating antibiotics, antitumorantibiotics (e.g., trastuzumab), binding epidermal growth factorreceptors (tyrosine-kinase inhibitors), aromatase inhibitors,anti-metabolites (e.g., folic acid analogs, methotrexate,5-fluoruracil), mitotic inhibitors (e.g., taxol, paclitaxel, docetaxel),growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomeraseinhibitors, biological response modifiers, anti-hormones,anti-androgens, and various cytokines for immunotherapy. Representativecytotoxic agents include BCNU, cisplatin, gemcitabine, hydroxyurea,paclitaxel, temozomide, topotecan, fluorouracil, vincristine,vinblastine, procarbazine, dacarbazine, altretamine, cisplatin,methotrexate, mercaptopurine, thioguanine, fludarabine phosphate,cladribine, pentostatin, fluorouracil, cytarabine, azacitidine,vinblastine, vincristine, etoposide, teniposide, irinotecan, docetaxel,doxorubicin, daunorubicin, dactinomycin, idarubicin, plicamycin,mitomycin, bleomycin, tamoxifen, flutamide, leuprolide, goserelin,aminoglutethimide, anastrozole, amsacrine, asparaginase, mitoxantrone,mitotane, and amifostine.

Suitable therapeutic drugs include siRNAs and antitumor tumor drugs thanfunction in cytoplasm.

In one embodiment, the invention provides a nanoparticle, comprising:

(a) a core comprising a magnetic material and having a surface;

(b) a polyarginine coating covalently coupled to the surface; and

(c) a therapeutic agent.

For this embodiment, suitable therapeutic agents are as described above.

For embodiments of the invention that include therapeutic agents, thetherapeutic agent can be covalently coupled to the nanoparticle ornon-covalently (e.g., ionic) associated with the nanoparticle. Fortherapeutic agent delivery, the therapeutic agent can be covalentlylinked (e.g., through a cleavable linkage) or physically adsorbed to(e.g., electrostatic or van der Waals interactions) to the nanoparticle,or embedded within the nanoparticle's coating.

In certain embodiments, the therapeutic agent is covalently coupled tothe nanoparticle through a cleavable linkage. Suitable cleavablelinkages include linkages cleavable under acidic conditions such as inthe acidic microenvironment of cancer cell (e.g., pH less thanphysiological pH, from about 4.0 to about 6.8. Representative cleavablelinkages include acetal, hydrazone, orthoester, and thioester linkages.

For embodiments of the nanoparticle that include a nucleic acidtherapeutic agent (e.g., siRNA), the nanoparticle is a nanovector. Forthese embodiments, the terms “nanoparticle” and “nanovector” are usedinterchangeably.

Targeting Agents.

Suitable targeting agents include compounds and molecules that directthe nanoparticle to the site of interest. Suitable targeting agentsinclude tumor targeting agents. Representative targeting agents includesmall molecules, peptides, proteins, aptamers, and nucleic acids.Representative small molecule targeting agents include folic acid andmethotrexate (folate receptors), non-peptidic RGD mimetics, vitamins,and hormones. Representative peptide targeting agents include RGD (avβ3integrin), chlorotoxin (MMP2), and VHPNKK (endothelial vascular adhesionmolecules). Representative protein targeting agents include antibodiesagainst the surface receptors of tumor cells, such as monoclonalantibody A7 (colorectal carcinoma), herceptin (Her2/ner), rituxan (CD20antigen), and ligands such as annexin V (phosphatidylserine) andtransferrin (transferrin receptor). Representative aptamer targetingagents include A10 RNA apatamer (prostate-specific membrane antigen) andThrm-A and Thrm-B DNA aptamers (human alpha-thrombin protein). Targetsfor the agents noted above are in parentheses. Representative nucleicacid targeting agents include DNAs (e.g., cDNA) and RNAs (e.g., siRNA).

In one embodiment, the invention provides a nanoparticle, comprising:

(a) a core comprising a magnetic material and having a surface;

(b) a polyarginine coating covalently coupled to the surface; and

(c) a targeting agent.

In another embodiment, the invention provides a nanoparticle,comprising:

(a) a core comprising a magnetic material and having a surface;

(b) a polyarginine coating covalently coupled to the surface;

(c) a targeting agent; and

(d) a therapeutic agent.

For these embodiments, suitable targeting agents and therapeutic agentsare as described above.

Diagnostic Agents.

Suitable diagnostic agents include optical agents, such as fluorescentagents that emit light in the visible and near-infrared (e.g.,fluorescein and cyanine derivatives). Representative fluorescent agentsinclude fluorescein, OREGON GREEN 488, ALEXA FLUOR 555, ALEXA FLUOR 647,ALEXA FLUOR 680, Cy5, Cy5.5, and Cy7.

In one embodiment, the invention provides a nanoparticle, comprising:

(a) a core comprising a magnetic material and having a surface;

(b) a polyarginine coating covalently coupled to the surface; and

(c) a diagnostic agent.

In another embodiment, the invention provides a nanoparticle,comprising:

(a) a core comprising a magnetic material and having a surface;

(b) a polyarginine coating covalently coupled to the surface;

(c) a diagnostic agent; and

(d) a therapeutic agent.

In a further embodiment, the invention provides a nanoparticle,comprising:

(a) a core comprising a magnetic material and having a surface;

(b) a polyarginine coating covalently coupled to the surface;

(c) a diagnostic agent;

(d) a targeting agent; and

(e) a therapeutic agent.

For these embodiments, suitable diagnostic agents, therapeutic agents,and targeting agents are as described above.

The preparation of representative nanoparticles of the invention isdescribed in Example 1 and illustrated schematically in FIGS. 1A-1D. Aschematic illustration of a representative nanoparticle of the inventionincluding a therapeutic agent (e.g., siRNA) and a fluorescent agent(e.g., DY547) is shown in FIG. 1D.

In another aspect of the invention, a composition is provided thatincludes a nanoparticle of the invention and a carrier suitable foradministration to a warm-blooded subject (e.g., a human subject).Suitable carriers include those suitable for intravenous injection(e.g., saline or dextrose).

Methods for Using Nanoparticle Having Polyarginine Coating

In other aspects, the invention provides methods for using thenanoparticles of the invention.

In certain embodiments, the invention provides methods for introducing amaterial (e.g., therapeutic and/or diagnostic agent) to a cell. In otherembodiments, the invention provides imaging methods such as magneticresonance imaging when the core has magnetic resonance activity, andoptical imaging when the nanoparticle includes a fluorescent agent. Asnoted above, the nanoparticles of the invention can also be used fordrug delivery when the nanoparticle includes a therapeutic agent. Fornanoparticles of the invention that include targeting agents, imaging ofand drug delivery to target sites of interest are provided.

In one embodiment, the invention provides a method for introducing ananovector into a cell via transcytosis. In the method, a cell iscontacted with a nanoparticle of the invention. As noted herein, thenanoparticles of the invention advantageously utilize transcytosisrather than endocytosis for transport into a cell. The ability to use acell transcytosis mechanism renders the nanoparticles of the invention(e.g., nanovectors) particularly well suited for the intracellulardelivery of cargo (e.g., therapeutic agents such as siRNA).

In another embodiment, the invention provides a method for silencing orreducing the expression level of a gene. In the method, a cell ofinterest is contacted with a nanoparticle of the invention in which thenanoparticle comprises a vector (i.e., suitable siRNA) effective tosilence or reduce the expression level of the particular gene.

In a further embodiment, the invention provides a method for detecting(or imaging) cells or tissues by magnetic resonance imaging, comprising:

(a) contacting cells or tissues of interest with a nanoparticle of theinvention having affinity and specificity for the cells or tissues ofinterest, wherein the nanoparticle comprises

-   -   (i) a core comprising a magnetic material and having a surface,    -   (ii) a polyarginine coating covalently coupled to the surface,        and    -   (iii) a targeting agent, wherein the targeting agent has an        affinity and specificity to the cells or tissues of interest;        and

(b) measuring the level of binding of the nanoparticle to the cells ortissues of interest, wherein an elevated level of binding, relative tonormal cells or tissues, is indicative of binding to the cells ortissues of interest.

In the method, the level of binding is measured by magnetic resonanceimaging techniques. In a further embodiment of the above method, thenanoparticle further includes a fluorescent agent. In this embodiment,the level of binding can be measured by magnetic resonance and/orfluorescence imaging techniques. The methods are applicable to detectingor imaging cells or tissues in vitro. The methods are also applicable todetecting or imaging cells or tissues in vivo. In this embodiment, thenanoparticles are administered to a subject (e.g., warm-blooded animal)by, for example, intravenous injection.

In another embodiment, the invention provides a method for treating atissue, comprising contacting a tissue of interest with a nanoparticleof the invention having affinity and specificity for the tissue ofinterest, wherein the nanoparticle comprises

(a) a core comprising a core material and having a surface,

(b) a polyarginine coating covalently coupled to the surface, and

(c) a targeting agent, wherein the targeting agent has an affinity andspecificity to the cells or tissues of interest.

In a further embodiment of the above method, the nanoparticle furthercomprises a therapeutic agent. In this embodiment, the therapeutic agentcan be covalently linked (e.g., through a cleavable linkage) orphysically adsorbed to (e.g., electrostatic or van der Waalsinteractions) the nanoparticle, or embedded within the nanoparticle'scoating. The methods are applicable to treating tissues in vitro. Themethods are also applicable to treating tissues in vivo. In thisembodiment, the nanoparticles are administered to a subject (e.g.,warm-blooded animal) by, for example, intravenous injection.

The following is a description of specific nanovectors of the inventionand methods of their use.

Magnetic nanoparticles (MNPs) coated with pArg and functionalized withsiRNA and their gene-silencing efficiency in tumor cell lines of thebrain, breast, and prostate were evaluated. MNPs coated with polylysine(pLys) or PEI were also prepared for comparison. pLys is anothercommonly used cationic polypeptide to complex siRNA and deliver siRNA tocytoplasm of cells via endocytosis and endosomal escape.

Nanovector Synthesis

FIGS. 1A-1D illustrates the fabrication scheme a representativenanoparticle of the invention involving the covalent attachment ofcationic polymers and siRNA to the amine-functionalized MNPs. The MNPconsists of a 10-12 nm iron oxide core coated with siloxane to which a44-mer of PEG-amine is anchored (FIG. 1A). The MNP was then modifiedwith 10-kDa cationic polymers (pLys, pArg, or PEI) to produce NP-pLys,NP-pArg and NP-PEI using the conjugation scheme in FIG. 1C. The chemicalstructures of the cationic polymers are shown in FIG. 1B.

To assess the gene delivery efficacy, Cy5 fluorescently labeled, thiolmodified siRNA (21 base-pairs, 5.7 nm length) designed to silence greenfluorescence protein (GFP) transgene expression was covalently attachedto the functional amine groups on the surface of NP-pLys, NP-pArg andNP-PEI using the heterobifunctional linker SIA (FIG. 1D) to formNP-pLys-siRNA, NP-pArg-siRNA and NP-PEI-siRNA.

This non-labile covalent attachment of siRNA to a nanocarrier ispreferable for in vivo applications because it ensures that thesiRNA-carrier construct will remain intact during blood circulation.Furthermore, this conjugation strategy does not compromise the knockdownefficiency of the siRNA. To favor thioether bond formation overelectrostatic binding of the negatively charged siRNA with cationic NP,the SIA reaction was performed in a high ionic strength buffer.

Nanovector Physicochemical Characterization

The successful immobilization of cationic polymers onto NP was verifiedusing proton NMR (¹H-NMR). Spectra from base MNPs, relevant constituentpolymers, and the three different nanovector formulations confirm thesuccessful immobilization of their expected polymer coatings FIGS. 2Aand 2B. PEG on NP was identified at 3.7 ppm corresponding to the protonsof the ethylene unit. PEI was identified at 3.75-3 ppm corresponding tothe protons of its ethylene units. pLys was identified at 3, 1.75, and1.45 ppm corresponding to the protons of the ε, δ and β, and γ carbonsof the lysine side chain. pArg was identified at 3.25, 1.8, and 1.6 ppmcorresponding to the protons of the δ, β, and γ carbons of the arginineside chain, respectively.

siRNA loading onto the nanovector was assessed using gel retardationassays where NP bound siRNA would not migrate down the gel.NP-pLys-siRNA, NP-PEI-siRNA and NP-pArg-siRNA were prepared at 20:1nanoparticle:siRNA weight ratios (Fe mass of NP:siRNA mass). Withoutpurification, the reaction products were loaded into agarose gels andunbound siRNA (bottom of gel) was separated from MNP-bound siRNA throughelectrophoresis. Each NP formulation showed complete binding of siRNA asno unbound siRNA was observed in the gels (FIG. 3A). A similar migrationprofile is demonstrated in the heparin treated samples (FIG. 3A), whichdisrupts electrostatic interactions, confirming successful covalentattachment of siRNA to MNP for all three nanovector formulations.

The hydrodynamic size of each nanovector formulation before and aftersiRNA loading was measured by dynamic light scattering (DLS) (FIG. 3B).Z-average diameters of the nanovectors were 53.4 nm for NP-pLys-siRNA,69.4 nm for NP-PEI-siRNA and 53.5 nm for NP-pArg-siRNA. No significantchange in hydrodynamic size was observed after siRNA attachment tonanovectors, reflecting their stability. Notably, the hydrodynamic sizefalls within the acceptable size range (5<d<200 nm) that facilitates invivo navigation and evasion of sequestration by macrophages.

TEM images of three nanovector formulations show no signs of aggregation(FIG. 3D, top row). Higher magnification images in FIG. 3D (bottom row)reveal a lower density shell surrounding the MNP cores, which is absenton the base MNPs (FIG. 9). This shell structure further confirms thecationic polymer coating on the MNPs.

The zeta potential of each nanovector formulation was measured at pH 7.4in 10 mM HEPES buffer by DLS. All three nanovector formulations werehighly cationic prior to siRNA attachment, with zeta potentials of 22 mVfor NP-pLys, 39 mV for NP-PEI and 21 mV for NP-pArg (FIG. 3C). AftersiRNA attachment, the zeta potentials of all formulations droppedsignificantly confirming successful siRNA conjugation. However,NP-PEI-siRNA remained highly cationic (30 mV) while the NP-pLys-siRNA(0.5 mV), and NP-pArg-siRNA (2.5 mV) displayed near-neutral zetapotentials under physiological pH conditions. A neutral NP formulationgenerally has better biocompatibility and prolonged circulating time inblood than positively charged formulations.

The three various nanovector formulations were similar in sizes, but haddifferent zeta potentials. They differ in composition and abundance ofsurface amino groups, providing us an opportunity to compare theinfluence of these key parameters on siRNA delivery and cytotoxicity.The charge and composition of the cationic polymer coating regulates thebuffering capacity and proton sponge behavior of the nanovector. Toillustrate this, nanovectors were titrated with HCL and the change in pHwas monitored. Resistance to pH change indicates an increase in protonabsorption of the nanovector. The buffering capacity was seen toincrease in the order of NP-pArg-siRNA<NP-pLys-siRNA<NP-PEI-siRNA (FIG.4A). This trend minors the buffering capacities of the coating polymersalone (FIG. 4B), indicating they were not affected by attachment to NPsor to siRNA.

To ensure the shell structure did not compromise the magnetic propertiesof the nanovectors, their magnetic relaxivities on MRI were evaluated.All nanovector formulations showed higher R₂ relaxivities compared tothe base NP (FIGS. 5A and 5B), suggesting that the attachment ofcationic polymers and siRNA to MNPs did not compromise, but rather,improved the magnetic properties. This improvement is likely due to theimproved water absorption at the surface of the core MNP due to thepresence of the hydrophilic cationic polymers.

Nanovector Internalization, Gene Knockdown and Monitoring of Toxicity

The nanovectors were evaluated in vitro for siRNA delivery and genesilencing in three cancer cell lines (C6/GFP⁺ brain, MCF7/GFP⁺ breast,and TC2/GFP⁺ prostate) that stably express the GFP gene. First, theamount of siRNA delivered to cells by each nanovector formulation wasevaluated using the fluorescently labeled siRNA (FIGS. 6A-6C). All threenanovectors delivered similar amounts of siRNA to C6/GFP⁺ cells (FIG.6A). Conversely, NP-pArg-siRNA delivered a markedly higher quantity ofsiRNA to MCF7/GFP⁺ cells than NP-pLys-siRNA and NP-PEI-siRNA (FIG. 6B).In TC2/GFP⁺ cells, both NP-pArg-siRNA and NP-pLys-siRNA delivered moresiRNA than NP-PEI-siRNA. These data demonstrate that the pArg coatingwas most broadly effective in facilitating the delivery of siRNA-loadednanovectors to target cells.

The influence of nanovector treatments on cell viability was evaluatedusing Alamar blue assays. Cells were treated with each nanovectorformulation and a commercial transfection reagent Dharmafect 4(Dharmacon Inc.) as a reference. The NP-pArg-siRNA treatment was theleast toxic to C6/GFP⁺ cells (90% viability (% V)), with NP-pLys-siRNA(70.6% V) and NP-PEI-siRNA (78.9% V) most toxic (FIG. 6D). A similartrend in viability was observed with MCF7/GFP⁺ cells (FIG. 6E) whereNP-pArg-siRNA treatment produced the least cytotoxicity (91.2% V),similar to Dharmafect 4 treatment (89% V) and much lower thanNP-pLys-siRNA (70.2% V) and NP-PEI-siRNA (49% V) treatments. This trendwas again observed in TC2/GFP⁺ cells (FIG. 6F). Both NP-pArg-siRNA(104.3% V) and Dharmafect 4 (92.5% V) treatments were well tolerated,while the NP-pLys-siRNA (77.5% V) and NP-PEI-siRNA (78.7% V) treatmentswere both moderately toxic.

The GFP gene-silencing efficacy of each nanovector formulation was thenevaluated (FIGS. 6G-6I). The NP-pArg-siRNA treatment was most effectivein gene silencing in C6/GFP⁺ cells (52.9% GFP knockdown (% GKD)),whereas Dharmafect 4 (36.4% GKD), NP-pLys-siRNA (22.7% GKD) andNP-PEI-siRNA (6.8% GKD) were all significantly less effective (FIG. 6G).A similar trend was observed in MCF7/GFP⁺ cells (FIG. 6H). NP-pArg-siRNAshowed the highest potency for silencing (68.2% GKD), whereas Dharmafect4 (51.3% GKD), NP-pLys-siRNA (39.1% GKD) and NP-PEI-siRNA (32.8% GKD)treatments were all less effective. In TC2/GFP⁺ cells it was againobserved that NP-pArg-siRNA (24% GKD) was most effective in genesilencing compared to the other two different nanovector formulations,NP-pLys-siRNA (13.9% GKD) and NP-PEI-siRNA (13.4% GKD) (FIG. 6I). Inthis cell line Dharmafect 4 (31.3% GKD) was slightly more effective thanNP-pArg-siRNA.

To further confirm the siRNA delivery and gene knockdown achieved byNP-pArg-siRNA to the three types of cells (C6/GFP+, MCF7/GFP+, andTC2/GFP+) confocal fluorescence microscopy was performed. Shown in FIGS.7A-7C are fluorescence images of untreated and NP-pArg-siRNA treatedC6/GFP+ (FIG. 7A), MCF7/GFP+ (FIG. 7B), and TC2/GFP+ (FIG. 7C) cells. Inall images cell nuclei were stained with DAPI (blue) and membranes withWGA-647 (green). Treatments with NP-pArg-siRNA were administered asdescribed above at a concentration of 1.2 μg of siRNA/ml. As shown inall three-cell lines tested internalized siRNA (red, second column) canbe visualized in cells treated with NP-pArg-siRNA. The overlay images(third column) reveal that the delivered siRNA molecules arepredominantly localized in the perinuclear region of cells, the regionof cell where siRNA molecules are recognized by the RISC complex. Thisobservation confirms the proper trafficking of siRNA within cells. TheGFP expression analysis (light green, fourth column) showed that theNP-pArg-siRNA treatment reduced the GFP expression of cells in all celllines compared to the untreated cells.

Based on the transfection and cell viability studies, NP-pArg-siRNA hasthe favorable overall properties as a theranostic agent compared toother nanovector formulations studied. This might be attributed to itsneutral zeta potential and limited proton sponge effect, which minimizesthe potential deleterious effects caused by non-specific interactionswith anionic intracellular components (e.g., mRNA, DNA). Thearginine-rich nanoparticle may travel across cell membranes throughtranscytosis without being trapped in endosome vesicles. NP-pArg-siRNAmay be similarly trafficked into cells accounting for its high genesilencing efficiency despite limited proton sponge capacity.

Nanovector Intracellular Trafficking

To determine the uptake mechanism of each nanovector formulation,C6/GFP⁺ cells treated with nanovectors were imaged with TEM (FIGS. 8Aand 8B). NP-pLys-siRNA and NP-PEI-siRNA could be observed entering thecell through endocytic pathways, whereas NP-pArg-siRNA appeared totranscytose across the cell membrane in the absence of independentendocytic engulfment (FIG. 8A). Furthermore, once inside the cellNP-pLys-siRNA and NP-PEI-siRNA could be seen escaping endosomal vesicleswhile NP-pArg-siRNA were free in the cytoplasm with no visible endosomalvesicle (FIG. 8B). These TEM images provide an explanation as to why theNP-pArg-siRNA formulation appeared efficient in inducing gene knockdown(FIGS. 6A-6I), despite its limited buffering capacity (FIG. 4A). TheNP-pArg-formulation does not require endosome escape properties owing toits ability to completely bypass the endocytosis pathway. Conversely,the NP-pLys-siRNA and NP-PEI-siRNA formulations must escape endosomalvesicles to localize in the cytoplasm.

The present invention provides nanoparticles useful as siRNA carriersthat utilize the polyarginine (pArg) peptide as a coating material. pArgis a naturally occurring, biodegradable polypeptide that offers improvedbiocompatibility (decreased toxicity) over PEI and PAMAM. Furthermore,because cell transcytosing proteins are known to avoid endosomalcompartmentalization and are commonly found with arginine-rich domains,the pArg-coated nanovectors of the invention offer the advantage oftranscytosing ability and thus offer more efficient siRNA delivery withgreatly improved biocompatibility over NPs coated with syntheticpolymers such as PEI.

As used herein, the term “about” refers to values +/−5% of the recitedvalue.

The following is provided for the purpose of illustrating, not limiting,the invention.

The Preparation, Characterization, and Properties of RepresentativePolyarginine-Coated Nanoparticles (NP-pArg and NP-pArg-siRNA)

The following is a description of the preparation and characterizationof representative arginine-coated nanoparticles of the invention(NP-Arg). The preparation and characterization of representativearginine-coated nanoparticles conjugated with siRNA (NP-pArg-siRNA) arealso described. The particles and their preparation are schematicallyillustrated in FIGS. 1A-1D.

A10 reagents were purchased from Sigma Aldrich (St. Louis, Mo.) unlessotherwise specified.

Amine-Terminated PEG-Coated Nanoparticles

Amine-terminated PEG-coated iron oxide nanoparticles (NPs) with a 12 nmcore diameter were synthesized as described in Fang C, Bhattarai N, SunC, Zhang MQ. Functionalized Nanoparticles with Long-Term Stability inBiological Media. Small. 2009; 5:1637-41, expressly incorporated hereinby reference in its entirety. A schematic illustration of thepreparation of representative amine-terminated PEG-coated iron oxidenanoparticles (NP-SAS-PEG-NH₂) is shown in FIG. 10.

Oleic acid-coated nanoparticles (NP-OA) were synthesized via thermaldecomposition of iron oleate complex. To render the nanoparticleshydrophilic, the NP-OAs were reacted with triethoxysilylpropylsuccinicanhydride (SAS) to form SAS-coated nanoparticles (NP-SAS) via aligand-exchange and condensation process. Amine-functionalized PEG wasattached to NP-SAS via N,N′-dicyclohexylcarbodiimide (DCC) mediatedcoupling reaction to yield PEGylated nanoparticles (NP-SAS-PEG-NH₂).NP-SAS-PEG-NH₂ bears amine groups at the free termini of PEG chainsallowing for further conjugation with bioactive molecules or ligands.The PEG coating also serves to prevent nanoparticles from agglomerationand protein adsorption.

Synthesis of NP-SAS-PEG-NH₂.

To a 5 ml of toluene solution containing 50 mg (iron content) of NP-OA,40 mL of acetone was added, and the nanoparticles were collected bycentrifugation. The nanoparticles were redispersed in 50 mL anhydroustoluene and transferred to a three-neck flask equipped with a heater.After the system was sealed and purged with nitrogen, 0.15 mL of SAS wasinjected, and the solution was heated to 100° C. for 12 hours. Thenanoparticles were precipitated by the addition of hexane, and collectedusing a rare earth magnet. The nanoparticles were washed twice withhexane and redispersed in anhydrous tetrahydrofuran (THF). To thissolution, 100 mg of H₂N-PEG-NH₂ and 2.5 mg ofN,N′-dicyclohexylcarbodiimide (DCC) were added, and the reaction mixturewas sonicated in a sonication bath for 12 hrs at 25° C. Thenanoparticles was precipitated by the addition of 200 mL of hexane, andcollected using a rare earth magnet. The precipitated nanoparticles wereredispersed in 50 mL of anhydrous THF, and 250 mg of PEG-bis(amine) and12.5 mg of DCC were added. The reaction mixture was kept in a sonicationbath for 16 hrs at 25° C. The resulting product was precipitated by theaddition of hexane, and collected using a rare earth magnet. After twoadditional cycles of THF-redispersion and ether-precipitation, theresidue solvent was evaporated and the nanoparticles were redispersed in5 mL PBS. After passing through a 0.2 μm syringe filter, thenanoparticles were purified through gel filtration chromatography bySephacryl S-200 column. The nanoparticles were stored in 0.1 M sodiumbicarbonate buffer (pH 8.5). The concentration of nanoparticles wasdetermined by inductively coupled plasma atomic emission spectroscopy(ICP-AES).

TEM images showed that both NP-OA and NP-SAS-PEG-NH₂ were spherical andwell dispersed, with a core size of about 12 nm. No aggregation ofNP-SAS-PEG-NH₂ was observed, indicating that no inter-particlecrosslinking occurred during the ligand exchange and PEG modification.Nanoparticle surface modification was confirmed by FTIR and XPS. The IRspectra of oleic acid coated nanoparticles exhibit the characteristicC—H stretch bands of methyl and methylene groups at 2930 and 2849 cm⁻¹and surface-complexed carbonyl stretch peaks at 1553 and 1433 cm⁻¹.After the surface modification of nanoparticles with SAS, the Si—O—Rvibrational bands, including a broad peak around 1031 cm⁻¹ and a minorpeak at 1188 cm⁻¹, were observed indicating the formation of complexsiloxane bonds. The relative intensity of surface-complexed carbonylbands increased, compared to oleic acid-coated NPs, indicative ofsuccessful SAS attachment. The absence of characteristic anhydride peaksat 1850-1800 cm⁻¹ and 1790-1740 cm⁻¹, suggesting that these groups wereeither hydrolyzed or bonded to the iron oxide surface. After the surfacemodification of NP-SAS with amine-functionalized PEG, multiple bands at1458, 1346, 1244, 1112 and 949 cm⁻¹ were observed, corresponding to thedifferent vibrational modes of PEG's C—O—C bonds. The bands at 1642 and1559 cm⁻¹ can be assigned to either primary amine groups ormono-substituted amide, indicating the successful covalent attachment ofPEG on the free carboxyl groups of NP-SAS. The number of reactive aminegroups on nanoparticles by quantifying pyridine-2-thione followingreaction with N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP). Thenumber of amine groups on each nanoparticle was about 70. As the numberof amine groups is proportional to the number of PEG chains, this resultalso suggests the formation of a high density PEG coating on the surfaceof the nanoparticle.

The hydrodynamic sizes of NP-OA in toluene and NP-SAS-PEG-NH₂ inphosphate-buffered saline (PBS) were determined to be 18 nm and 38 nm,respectively, by dynamic light scattering. The size increase (about 20nm) of the NP-SAS-PEG-NH₂ compared to NP-OA can be attributed to the PEG(MW=2,000) coating and the water molecules associated to PEG.

NP-pLys, NP-pArg, and NP-PEI Synthesis

The cationic polymer coated nanoparticles were prepared fromamine-terminated PEG-coated iron oxide nanoparticles (NP in FIG. 1A orNP-SAS-PEG-NH₂ in FIG. 10). NPs were modified with cationic polymersthrough the formation of a thioether bond between thiolated polymers andNPs activated with iodoacetyl groups. Specifically, 10.5 mg of NP in 1mL of thiolation buffer (0.1M sodium bicarbonate, 5 mM EDTA, pH 8.0) wasreacted with 10.5 mg succinimidyl iodoacetate (SIA, MolecularBiosciences, Boulder, Colo.) in 112.5 μl DMSO. Concurrently, 15.75 mg ofpoly-lysine (MW 10,000), poly-arginine (MW 10,000), or PEI (MW 10,000)were dissolved in 1.725 ml thiolation buffer and modified with 26 μl ofa 10 mg/ml Traut's reagent (2-iminothiolane, Molecular Biosciences,Boulder, Colo.). All of the reactions were protected from light andensued under gentle rocking. After 2 hours, excess SIA was removed fromNP-SIA using a pre-packed PD-10 desalting column (GE Healthcare,Piscataway, N.J.) equilibrated with thiolation buffer. Uponpurification, the NPs were split into three equal volumes, eachcontaining 3.5 mg Fe, and added to the three polymer solutions forreaction. The reaction were protected from light and ensued for 2 hourswith gentle rocking and was then stored at 4° C. overnight. Thereactants for each reaction were then purified to remove excess polymerusing columns packed with S-200 Sephacryl resin (GE Healthcare,Piscataway, N.J.) equilibrated with thiolation buffer.

siRNA Preparation

siRNA sequences designed to knockdown GFP expression:5′-GCAAGCUGACCCUGAAGUUCUU-3′-antisense (SEQ ID NO: 1) and5′-GAACUUCAGGGUCAGCUUGCUU-3′-sense (SEQ ID NO: 2) were purchased fromIntegrated DNA Technologies, Inc. (IDT, San Diego Calif.). Thesesequences were acquired with protected-thiol modifications on the 5′ endof the sense strand and with Cy5 modification on the 5′ end of theantisense strand. siRNA sequences were received as single strands andwere annealed to their complementary strand in annealing buffer (12 mMpotassium chloride, 1.2 mM HEPES, 0.04 mM magnesium chloride, pH 7.5) byincubating at 95° C. for five minutes, then 37° C. for 1 hr, and thenstored at −20° C.

NP-pLys-siRNA, NP-pArg-siRNA, and NP-PEI-siRNA Synthesis

NP-pLys, NP-pArg, and NP-PEI were modified with SIA at a 5:1 weightratio of Fe:SIA. The SIA was dissolved in DMSO such that the finalreaction volume was 10% DMSO. The reactions were protected from lightand ensued for 2 hours with gentle rocking. Meanwhile, a 57.73 mg/mlTCEP (tris (2-carboxyethyl) phosphine hydrochloride) solution wasprepared. The TCEP and GFP siRNA were combined at a 1:1 volume ratio,protected from light, and allowed to react for 1 hr with gentle rocking.The resultant siRNA was purified using a 2 ml Zeba column (Thermo FisherScientific, Waltham, Mass.) equilibrated with thiolation buffer. Thenanoparticles were purified using pre packed PD-10 columns equilibratedwith thiolation buffer. Upon purification, the siRNA was added to eachof the three types of NPs at a 20:1 Fe:siRNA weight ratio. The reactionswere protected from light and allowed to proceed for 2 hrs with gentlerocking and the resultant nanoparticle:siRNA nanovectors were usedwithout any further purification.

Proton NMR Analysis of Cationic Polymer Attachment to NP

Each NP formulation (50 μg Fe) was dissolved in 50 μL of DCl and dilutedto 1 mL in D₂O, Similarly, cationic polymers were dissolved in 1 mL D₂Ocontaining 50 μL of DCl. Proton NMR spectra were obtained on a BrukerAVance series spectrometer operating at 300 MHz.

Gel Retardation Assay

Attachment of siRNA to NPs was assessed using gel retardation assays. A4% low melting point agarose gel was prepared with 0.05 mg/mL ethidiumbromide. While maintaining a uniform concentration of siRNA, samples ofnanoparticle:siRNA complexes were prepared at a weight ratio of 20:1 (Femass of NP: siRNA mass). Samples were either left untreated or treatedwith heparin (1000 units/ml, 10 mL heparin/mg siRNA), and incubated for30 min at room temperature to block the electrostatic interactionbetween the nanoparticles and siRNA. siRNA binding was analyzed by gelelectrophoresis at 55 V for 90 min. Images were acquired on a Gel Doc XR(Bio-Rad Laboratories, Hercules, Calif.).

Nanovector Size and Zeta Potential Characterization

Hydrodynamic sizes and zeta potentials of the different nanoparticleformulations were analyzed at 100 μg/mL in 20 mM HEPES buffer (pH 7.4)using a DTS Zetasizer Nano (Malvern Instruments, Worcestershire, UK).

Evaluation of Nanovector Magnetic Properties by MRI

Nanovector formulations were diluted in PBS, and then mixed with 25 μLof 1% agarose and loaded into an agarose gel phantom block. T2relaxation measurements were performed on a 4.7-T Bruker magnet (BrukerMedical Systems, Karlsruhe, Germany) equipped with Varian Inovaspectrometer (Varian, Inc., Palo Alto, Calif.). A 5 cm volume coil andspin-echo imaging sequence were used to acquire T2-weight images. Imageswere acquired using a repetition time (TR) of 3000 ms and echo times(TE) of 13.6, 20.0, 40.0, 60.0, 90.0 and 120.0 ms. The spatialresolution parameters were: acquisition matrix of 256×128, field-of-viewof 35×35 mm, section thickness of 1 mm and two averages. The T2 map wasgenerated by NIH ImageJ (Bethesda, Md.) based on the equation,SI=Aexp(−TE/T2)+B, where SI is the signal intensity, TE is the echotime, A is the amplitude, and B is the offset. The R2 map was generatedby taking the reciprocal of the T2 map.

Evaluation of Nanovector Buffering Capacity

NP-pLys-siRNA, NP-pArg-siRNA, and NP-PEI-siRNA were desalted usingpre-packed PD-10 columns equilibrated with DI water. 1 mg of Fe fromeach condition was then transferred to a 15 ml Falcon tube and broughtto pH 11.0 by addition of 0.1 N NaOH. The final volume for eachcondition was brought up to 2 ml with pH 11.0 DI water making the Feconcentration 500 μg/ml. The titration was performed with 2 μL additionsof 0.1 N HCl.

Cell Culture

C6 rat glioma (ATCC, Manassas, Va.) and MCF7 human adenocarcinoma (ATCC,Manassas, Va.) cells were maintained in Dulbecco's Modified Eagle Medium(DMEM, Invitrogen, Carlsbad, Calif.) supplemented with 10% FBS (AtlantaBiologicals, Lawrenceville, Ga.) and 1% antibiotic-antimycotic(Invitrogen, Carlsbad, Calif.) at 37° C. and 5% CO₂. Enhanced greenfluorescent protein (EGFP) expressing cells were produced by stablytransfecting cells with the pEGFP-N1 vector using the Effectenetransfection reagent (Qiagen, Valencia, Calif.) following themanufacturer's protocol. 48 hrs post-transfection, cells were sortedusing a FACS Vantage and maintained in fully supplemented DMEM with 1mg/ml G-418. TC2/GFP+ cells were maintained in Dulbecco's Modified EagleMedium (DMEM) (Invitrogen, Carlsbad, Calif.) supplemented with 10% FBS(Atlanta Biological, Lawrenceville, Ga.) and 1% antibiotic-antimycotic(Invitrogen, Carlsbad, Calif.) at 37° C. and 5% CO₂.

Cell Transfection

The day before transfection, cells were plated at 50,000 cells per wellin 24-well plates. For transfection of cells with nanovectorformulations, cells were treated with nanovectors for 8 hrs under normalgrowth conditions. After the 8-hour incubation the media were replacedand cells incubated for an additional 48 hrs before analyses.Transfections of cells with siRNA using Dharmafect 4 (Dharmacon,Lafayette, Colo.) were performed according to the manufacturer'sinstructions.

Cell Viability and Gene Silencing

Potential cytotoxicity associated with the nanovector formulations wasexamined by the Alamar blue assay. After treatment, cells were washedwith PBS three times, and incubated for 2 hrs with 10% Alamar blue(Invitrogen) in phenol red-free DMEM (supplemented with 10% FBS and 1%antibiotic-antimycotic). The percent reduction of Alamar blue wasdetermined following the manufacturer's protocol and used to calculatepercent viability of treated samples (untreated cells represent 100%viability).

To quantify the degree of GFP gene silencing, treated cells were washedwith PBS three times and lysed with 1% Triton X-100 in PBS. GFP proteinexpression was measured at an excitation and an emission wavelength of488 and 520 nm, respectively. GFP fluorescence levels were normalized tothe total number of viable cells, as determined by the Alamar Blueviability assay. Relative GFP expression levels were then calculatedbased on the reduction in GFP expression as compared to non-transfectedcells.

Confocal Fluorescence Microscopy

50,000 treated cells were plated on each of 24 mm glass cover slips andallowed to attach for 24 hrs. Cells were then washed with PBS and fixedin 4% formaldehyde (Polysciences Inc., Warrington, Pa.) for 30 min.Cells were then washed three times with PBS and membrane-stained withWGA-AF647 (Invitrogen, Carlsbad, Calif.) according to the manufacturer'sinstructions. Cover slips were then mounted on microscope slides usingProlong Gold antifade solution (Invitrogen, Carlsbad, Calif.) containingDAPI for cell nuclei staining. Images were acquired on a LSM 510 Metaconfocal fluorescence microscope (Carl Zeiss Inc., Peabody, Mass.) withthe appropriate filters.

Transmission Electron Microscopy (TEM)

One million C6 cells were seeded in 25 cm² flasks 24 hrs beforetreatment. Cells were then treated with nanovector formulations asdescribed for gene silencing experiments. Cells were then washed threetimes with PBS and incubated with ice cold Karnovsky's fixative for 24hrs. Following the fixation, the cells were processed directly fromflasks for sectioning. Cell sections were stained with osmium tetroxide,lead citrate, and uranyl acetate for TEM-contrast enhancement. Cellsamples were then imaged with a Philips CM100 TEM at 100 kV with a Gatan689 digital slow scan camera.

While illustrative embodiments have been illustrated and described, itwill be appreciated that various changes can be made therein withoutdeparting from the spirit and scope of the invention.

1. A nanoparticle, comprising: (a) a core having a surface andcomprising a core material; and (b) a polyarginine coating covalentlycoupled to the surface of the core.
 2. The nanoparticle of claim 1further comprising a therapeutic agent.
 3. The nanoparticle of claim 1further comprising a targeting agent.
 4. The nanoparticle of claim 1further comprising a therapeutic agent and a targeting agent.
 5. Thenanoparticle of claim 1, wherein the polyarginine has an averagemolecular weight from about 2,000 to about 200,000 g/mole.
 6. Thenanoparticle of claim 2, wherein the therapeutic agent is selected fromthe group consisting of a small organic molecule, a peptide, an aptamer,a protein, and a nucleic acid.
 7. The nanoparticle of claim 2, whereinthe therapeutic agent is an RNA or a DNA.
 8. The nanoparticle of claim2, wherein the therapeutic agent is an siRNA.
 9. The nanoparticle ofclaim 2, wherein the therapeutic agent is covalently coupled to thenanoparticle through a cleavable linkage.
 10. The nanoparticle of claim3, wherein the targeting agent is selected from the group consisting ofa small organic molecule, a peptide, an aptamer, a protein, and anucleic acid.
 11. The nanoparticle of claim 3, wherein the targetingagent is selected from the group consisting of a chlorotoxin, folicacid, methotrexate, RGD, VHPNKK, A10 RNA aptamer, transferrin, andherceptin.
 12. The nanoparticle of claim 1, wherein the core materialcomprises a material having magnetic resonance imaging activity.
 13. Thenanoparticle of claim 1 further comprising a poly(alkylene oxide)oligomer intermediate the core and the polyarginine coating.
 14. Thenanoparticle of claim 1, wherein the poly(alkylene oxide) oligomer iscovalently coupled to the core by siloxane linkages.
 15. Thenanoparticle of claim 2 further comprising a fluorescent agent.
 16. Thenanoparticle of claim 3 further comprising a fluorescent agent.
 17. Thenanoparticle of claim 4 further comprising a fluorescent agent.
 18. Thenanoparticle of claim 1 further comprising a fluorescent agent.
 19. Acomposition, comprising a nanoparticle of claim 1 and a carrier suitablefor administration to a warm-blooded subject. 20-24. (canceled)
 25. Amethod for introducing a nanovector into a cell via transcytosis,comprising contacting a cell with a nanoparticle of claim
 1. 26. Amethod for silencing or reducing the expression level of a gene,comprising contacting a cell of interest with a nanoparticle of claim 1,wherein the nanoparticle comprises a vector effective to silence orreduce the expression level of the gene.
 27. A method for detectingcells or tissues by magnetic resonance imaging, comprising: (a)contacting cells or tissues of interest with a nanoparticle of claim 1;and (b) measuring the level of binding of the nanoparticle, wherein anelevated level of binding, relative to normal cells or tissues, isindicative of binding to the cells or tissues of interest.
 28. A methodfor treating a tissue, comprising contacting a tissue of interest with ananoparticle of claim 1.